The synergistic effect of silicon-based substrates on the physical and chemical properties of aluminum nitride (AlN) thin films was investigated. AlN thin films were deposited by RF magnetron sputtering on Low Resistivity (LR) Si, Silicon On Insulator (SOI) and Si/SiO2 substrates at room temperature. The morphological and structural properties were investigated by X-ray diffraction (XRD), Raman spectroscopy, Atomic Force microscopy (AFM). XRD analyses evidenced the co-presence of (002) and (101) orientations. The substrate influence on films morphology, crystalline order, intrinsic stress and grain size is well evidenced, as shown by Raman and AFM analyses. These surface characterization techniques represent a valid support to select the suitable Si-substrate/piezoelectric thin film combination for the fabrication of a piezoelectric device. AlN sputtered on Si-LR substrate showed an enhancement of structural arrangement along (002) planes while the sample sputtered on Si/SiO2 resulted mainly oriented along (101) planes. For these reasons, further characterization was done: (002)-oriented AlN thin films were characterized in terms of piezoelectric response by piezometer and Piezoresponse Force Microscopy (PFM) measurements, while cytotoxicity and biocompatibility were investigated for (101)-oriented AlN thin films. This further investigation helped to assess the suitable film to integrate into piezoelectric devices operating in air or in liquid, respectively.